1. Field of the Invention
The invention relates to a DNA construct and a process for the fermentative production of fusion proteins using the construct.
2. Background Art
The market for recombinant protein pharmaceuticals (“Biologicals”) has grown enormously in recent years. The production costs for protein-based pharmaceutical active substances is still very high thereby providing an impetus for researchers to keep looking for more efficient, and therefore less expensive, processes and systems for their production. Protein producers that can be used include various microorganisms such as bacteria, yeasts, filamentous fungi, or else plant cells or animal cells. However, the Gram-negative enterobacterium Escherichia coli (E. coli) is currently the most frequently used organism for the production of recombinant proteins, owing to the fact that its genetics and physiology have been studied extensively, it has a short generation time and is simple to handle.
When producing recombinant proteins in E. coli, one frequently encounters the problem of inclusion body formation in the cell's cytoplasm. This difficulty can frequently be circumvented by designing the production system in such a way that the cell does not accumulate the recombinant protein to be prepared in each case (also referred to as the target protein as set forth below) in the cytoplasm, but actively secretes it into the periplasm and, ideally, even further into the culture medium. This can be achieved by the cell first synthesizing the target protein as a cytoplasmic precursor fusion protein (FIGS. 1 A and 1 B), the signal peptide (SP) being cleaved off in vivo during the secretion procedure into the periplasm, giving rise to the fusion protein (FIGS. 1 C and 1 D). In E. coli, the Sec system is responsible for the transport and the cleavage of the signal peptide. In principle, the extracellular production of target proteins makes use of signal peptides of periplasmic or extracellular proteins of E. coli. These E. coli proteins are secreted via the Sec pathway. Examples which may be mentioned here are the signal peptides of the E. coli proteins PhoA, OmpA, StII, Lpp and MalE.
In accordance with the Sec system, proteins are transported across the cytoplasmic membrane in the unfolded state and subsequently folded in the periplasm. In contrast, the TAT system (Twin Arginine Translocation) allows proteins to be secreted into the periplasm which have already undergone folding in the cytoplasm; however, this requires specific signal peptides (for example signal peptides of the proteins TorA or Tap). The review article by Choi and Lee (2004, Appl. Microbiol. Biotechnol. 64, 625-635) shows the state of the art on the secretory and extracellular production of recombinant proteins with reference to representative examples.
The accumulation of the target protein in the periplasm or in the culture medium has the following advantages over intracellular production: inter alia,    1) the N-terminal amino acid residue of the secreted target protein does not necessarily have to be methionine, but may be identical with the natural starting amino acid of the product,    2) the protease activity in the periplasm is markedly lower than in the cytoplasm,    3) the isolation of a protein from the periplasm or the culture medium is simpler than from the cytoplasm, since the former contain fewer contaminating host proteins, and    4) the formation of any disulfide bridges which may be required is made possible under the oxidative conditions of the periplasm.
In some cases, for example in the secretory production of recombinant proteins or peptides which are particularly sensitive to proteolytic degradation, or which tend to form inclusion bodies, it is necessary or, indeed, imperative for an effective production that the recombinant protein is not coupled directly to a signal peptide, but to the C terminus of a further protein, of the carrier protein (see FIG. 1 A: precursor fusion protein and 1 C: fusion protein). As a part of a secretable fusion protein, it is frequently possible to increase the solubility of the target protein in the fermentation medium. This may be achieved by coupling to a carrier protein followed by the rapid export of the complete precursor fusion protein from the cytoplasm may protect a target protein, or target peptide, which is sensitive to proteolysis from intracellular degradation. In order to protect a sensitive gene product from degradation, and thus to stabilize it, it is also possible to fuse the carrier protein with the C terminus of the target protein (see FIG. 1 B: precursor fusion protein and 1 D: fusion protein). An example for this is the protein glutathione S-transferase, which is used both as carrier protein and as dimerization domain in the secretion of a recombinant protease inhibitor as target protein (Tudyka and Skerra, 1997, Protein Science 6, 2180-2187).
A target protein as used herein means a recombinant protein, protein fragment or peptide that is to be produced extracellularly or periplasmically in yields of greater than 50 mg/l.
A precursor fusion protein as used herein means a protein consisting of a carrier protein and a target protein and a signal peptide (SP). Carrier protein and target protein are linked with one another by a sequence S which is enzymatically or chemically cleavable. The signal peptide is required for the translocation of the fusion protein across the cytoplasmic membrane. This signal peptide is cleaved from the fusion protein during the secretion process into the periplasm, giving rise to the fusion protein.
A fusion protein as used herein means a protein consisting of a carrier protein and a target protein. Carrier protein and target protein are linked with one another via a sequence S which is enzymatically or chemically cleavable.
A carrier protein as used herein means the portion of a precursor fusion protein, or of a fusion protein, which is used for stabilizing the target protein.
After the signal peptide has been cleaved from the precursor fusion protein, the fusion protein is accumulated in the periplasm or in the culture medium. The target protein is preferably linked to the carrier protein via an amino acid sequence, the cleavable sequence S (see FIG. 1), where the target protein can be cleaved off in vitro, either enzymatically or chemically, in a subsequent step, thereby being released.
A carrier protein that has been described as fusion partner for the extracellular production of proteins is, for example, the E. coli protein YebF (Zhang et al., 2006, Nature Biotechnology 24, 100-104 and WO2006017929A1).
The YebF protein has a primary structure of 118 amino acids and a molecular weight of 10.8 kDa and is currently the smallest carrier protein described for the heterologous expression of proteins in E. coli. A disadvantage in the use of the YebF protein as carrier protein is that the protein contains a total of three cysteine residues, two of which are located in the mature portion of the protein. These two cysteine residues can then form disulfide bridges with the target protein in the oxidizing medium of the periplasm, which may result in a misfolding of the target protein. In the most adverse case, this leads to an unusable target protein. The formation of incorrect disulfide bridges is possible both before and after the target protein has been cleaved from the YebF protein.
Another carrier protein is the OmpF protein of E. coli. Jeong and Lee describe the extracellular production of an OmpF-β-endorphin fusion protein with a derivative of the E. coli strain BL21 (DE3) (2002, Appl. Environ. Microbiol. 68, 4979-85). With a primary structure of 362 amino acids and a resulting molecular weight of 36 kDa, the OmpF carrier protein is considerably larger and heavier than the YebF carrier protein. This means that an E. coli cell, when producing a target protein, is metabolically much more stressed when OmpF is used as the carrier protein instead of YebF. This is particularly clear from the above paper by Jeong and Lee: a production of 5.6 g/l fusion protein only leads to 0.33 g/l β-endorphin.
Another carrier protein is the C-terminal fragment of the HlyA protein of E. coli. The hemolysin (HlyA protein) from E. coli is a pore-forming exotoxin with a molecular weight of 110 kDa, which is found in pathogenic E. coli strains causing, for example, infections of the urinary tract. The HlyA protein is transported out of the cell via the inner and the outer membranes via its own hemolysin transport system. The C-terminal domain of 218 amino acids of the protein, with a molecular weight of 23 kDa, is employed to produce the fusion proteins. This domain is required for the translocation across the two membranes. Human interleukin-6 can be produced extracellularly in the culture supernatant with the aid of an HlyA carrier protein. At 70 μg/l, the yields are extremely low however (Li et al., 2002, Gene 25, 437-447). This fragment, however, is unsuitable for the production of pharmaceutically active proteins, being the fragment of a toxic E. coli protein.
A further carrier protein for the extracellular production of target proteins is the eukaryotic protein hirudin from Hirodo medicinalis. Hirudin is a thrombin inhibitor, it binds to the protease thrombin with extremely high affinity (Ki=20 fM). The peptide, which consists of 65 amino acids, forms 3 internal disulfide bridges with its 6 cysteine residues. For the extracellular production of target proteins in E. coli, the carrier protein hirudin is fused with a bacterial signal peptide. Signal peptides used are, for example, sequences of the E. coli proteins PhoA or OmpC (EP1364029B1), or else the signal peptide of the cyclodextrin glycosyltransferase (EP0448093B1). Owing to its biological activity as thrombin inhibitor, hirudin is unsuitable for the production of pharmacological proteins since it must be ensured during the production process of a target protein that no contamination with the carrier protein hirudin is present. This makes the purification process of the target protein very complicated and therefore expensive.